The hyperthermophiles of archaebacteria are a recently discovered group of microorganisms that grow optimally at temperatures around 100.degree. C. Many species of these extremely thermophilic bacteria-like organisms have been isolated, mainly from shallow submarine and deep sea geothermal environments. Most of the archaebacteria are strict anaerobes and depend on the reduction of elemental sulfur for growth.
The "hyperthermophiles" are presently represented by three distinct genera, Pyrodictium, Pyrococcus, and Pyrobaculum. Pyrodictium brockii (T.sub.opt 105.degree. C.) is an obligate autotroph which obtains energy by reducing S.sup.0 to H.sub.2 S with H.sub.2, while Pyrobaculum islandicum (T.sub.opt 100.degree. C.) is a facultative heterotroph that uses either organic substrates or H.sub.2 to reduce S.sup.0. In contrast, Pyrococcus furiosus (T.sub.opt 100.degree. C.) grows by a fermentative-type metabolism rather than by S.sup.0 respiration. It is a strict heterotroph that utilizes both simple and complex carbohydrates where only H.sub.2 and CO.sub.2 are the detectable products. The organism reduces elemental sulfur to H.sub.2 S apparently as a form of detoxification since H.sub.2 inhibits growth.
The discovery of microorganisms growing optimally around 100.degree. C. has generated considerable interest in both academic and industrial communities. Both the organisms and their enzymes have the potential to bridge the gap between biochemical catalysis and many industrial chemical conversions. However, knowledge of the metabolism of the hyperthermophilic microorganisms is presently very limited.
The ligase chain reaction (LCR) provides a powerful method for the rapid and sensitive amplification of DNA fragments. LCR allows the specific detection of a target nucleic acid sequence with a single base mutation. LCR has facilitated the development of gene diagnostic technologies including the determination of allelic variation, and the detection of infectious and genetic disease disorders.
LCR is performed by repeated cycles of heat denaturation of a DNA template containing the target sequence, annealing a first set of two adjacent oligonucleotide probes to the target DNA sequence in a unique manner, and a second set of complementary oligonucleotide probes that hybridize to the sequence opposite to the target DNA sequence. Thereafter, a thermostable DNA ligase will covalently link each pair of adjacent probes provided there is complete complementarity at the junction of the two adjacent probes. Because the oligonucleotide products from one round may serve as substrates during the next round, the signal is amplified exponentially, analogous to the polymerase chain reaction (PCR).
LCR has been extensively described by Landegren et al., Science, 241:1077-1080 (1988); Wu et al., Genomics, 4:560-569 (1989); Barany, in PCR Methods and Applications, 1:5-16 (1991); and Barany, Proc. Natl. Acad. Sci. USA, 88:189-193 (1991).
An important aspect of successful LCR is to reduce background target-independent ligations, including blunt-end ligations. Such target-independent ligations produce a product the same size as the desired product from a target-directed LCR reaction, and as such are indistinguishable from the desired reaction product. The method requires a thermostable ligase to allow ligation to occur under temperature conditions that prevent mismatches from hybridizing to form acceptable substrates for a thermostable DNA ligase.
DNA ligases exhibiting limited temperature stability have been isolated from Thermus aguaticus (Taq), and from Thermus thermophilus (Tth). See, for example Takahashi et al., J. Biol. Chem., 259:10041-10047 (1984). However, these enzymes do not maintain thermostability at temperatures greater than about 65.degree. C. for prolonged periods of up to 10 to 30 minutes as required for typical LCR protocols. Thus, the known DNA ligases are unstable at high temperatures for prolonged periods, and therefore require a "pre-melt" step in LCR procedures to separate the two strands of the genomic DNA molecule prior to the addition of the enzyme followed by LCR cycles below about 85.degree. C. to 90.degree. C.
There continues to exist a need for a thermostable DNA ligase that can retain activity at high temperatures for prolonged periods of time, such as during ligase chain reactions.